Immobilized enzymes are utilized extensively in biosensor and related detection protocols in many technological applications. Immobilization generally offers improved enzyme stability and localization in comparison to soluble forms (Rojas-Melgarejo 2003). 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is the most common coupling chemistry used to couple proteins to many biomaterials. For example, to couple BMP-2 onto silk fibroin (Karageorgiou et al. 2004), to crosslink collagen and hyaluronic acid (Park et al. 2003) and collagen with glycosaminoglycan (Pieper et al. 2000). Covalent coupling of enzymes is necessary for gradient formation in order to further improve control of enzyme location and stability in comparison to more homogenous distributions currently studied. In particular, needs related to multidetection biosensor formats and gradients in responses and functions of such material would potentially advance technological utility of these materials.
To control protein patterning on surfaces there are several methods available. The most well-known is soft lithography (using PDMS as the material) such as microcontact printing (μCP) and micromolding in capillaries (MIMC). μCP is a method where a two-dimensional stamp creates complex patterns of proteins or biological materials (usually) on PDMS slabs. Using μCP, one substance can be printed at a time into complex shapes or self-assembled monolayers (Chiu et al. 2000) Park and Shuler 2003). μCP has also been used for tissue engineering applications. For example, laminin lanes were printed on biodegradable polyurethane, seeded with cardiomyocytes which grew to two-three cells layers thick in an attempt to create engineered tissue (McDevitt et al. 2003). MIMIC is another soft lithographic technique which uses microchannels formed by contacting the PDMS structure with a substrate. Desired fluid is flown to certain areas to form patterns (Park and Shuler 2003). The disadvantage of the two-dimensional flow through microchannels is that it is restricted to simple patterns. An extension of this method is a three-dimensional microfluidic channel system whereby complex and discontinuous patterns can be generated using more than one substance at a given time on a planar surface (Chiu et al. 2000). It would be interesting and useful if this method could be extended to create micro-patterned protein solutions in three-dimensional gels and foams. Nanoscale techniques, such as dip pen nanolithography, have also been used to pattern proteins such as collagen (Wilson et al., 2001). However, the scale and speed of such processes are limiting and the process is confined to two dimensional surfaces.
Current methods to develop protein gradients on surfaces are derived from the field of chemotaxis. The study of chemotaxis was accelerated by formation of non-linear gradients of soluble growth factors generated in the Boyden Chamber. However, the gradients generated were unstable and hence could not be used for a longer period of time (Boyden 1962). A microfluidic gradient generator was developed by Li Jeon et al. (2002) to make stable (stable until flow of is maintained), soluble and complex gradients of interleukin-8. However smearing of the gradient was observed and since the gradient was developed on two-dimensional PDMS slabs, it can only be extended to use in vitro and for shorter periods of time (Li Jeon et al. 2002). Accordingly, there is a need to develop 3D controlled immobilized protein gradient materials.